No abstract

RNase H1-dependent, phosphorothioate-modified antisense oligonucleotides (PS-ASOs) can enter cells through endocytic pathways and need to be released from the membrane-enclosed organelles, a limiting step for antisense activity. Accumulating evidence has suggested that productive PS-ASO release mainly occurs from late endosomes (LEs). However, how PS-ASOs escape from LEs is not well understood. Here, we report that upon PS-ASO incubation, COPII vesicles, normally involved in ER–Golgi transport, can re-locate to PS-ASO-containing LEs. Reduction of COPII coat proteins significantly decreased PS-ASO activity, without affecting the levels of PS-ASO uptake and early-to-late endosome transport, but caused slower PS-ASO release from LEs. COPII co-localization with PS-ASOs at LEs does not require de novo assembly of COPII at ER. Interestingly, reduction of STX5 and P115, proteins involved in tethering and fusion of COPII vesicles with Golgi membranes, impaired COPII re-localization to LEs and decreased PS-ASO activity. STX5 can re-locate to LEs upon PS-ASO incubation, can bind PS-ASOs, and the binding appears to be required for this pathway. Our study reveals a novel release pathway in which PS-ASO incubation causes LE re-localization of STX5, which mediates the recruitment of COPII vesicles to LEs to facilitate endosomal PS-ASO release, and identifies another key PS-ASO binding protein.

No abstract

Chemically modified antisense oligonucleotides (ASOs) with phosphorothioate (PS) linkages have been extensively studied as research and therapeutic agents. PS-ASOs can enter the cell and trigger cleavage of complementary RNA by RNase H1 even in the absence of transfection reagent. A number of cell surface proteins have been identified that bind PS-ASOs and mediate their cellular uptake; however, the mechanisms that lead to productive internalization of PS-ASOs are not well understood. Here, we characterized the interaction between PS-ASOs and epidermal growth factor receptor (EGFR). We found that PS-ASOs trafficked together with EGF and EGFR into clathrin-coated pit structures. Their co-localization was also observed at early endosomes and inside enlarged late endosomes. Reduction of EGFR decreased PS-ASO activity without affecting EGF-mediated signaling pathways and overexpression of EGFR increased PS-ASO activity in cells. Furthermore, reduction of EGFR delays PS-ASO trafficking from early to late endosomes. Thus, EGFR binds to PS-ASOs at the cell surface and mediates essential steps for active (productive) cellular uptake of PS-ASOs through its cargo-dependent trafficking processes which migrate PS-ASOs from early to late endosomes. This EGFR-mediated process can also serve as an additional model to better understand the mechanism of intracellular uptake and endosomal release of PS-ASOs.

Protein content was measured in zona‐free bovine oocytes and pre‐elongation stage embryos, following in vitro maturation, fertilisation, and then culture in Synthetic Oviduct Fluid medium supplemented with amino acids and 8 mg ml‐1 bovine serum albumin (BSA). Values (ng embryo‐1) of 122 ± 7.8, 137 ± 8.6, 111 ± 8.8, 115 ± 10.4, 139 ± 9.0 and 152 ± 10.1 were obtained for zona‐free mature oocytes, 2‐cell (day 2), 8‐cell (day 3), compact morula (day 6), blastocyst (day 7), and expanded blastocyst (day 8) stage embryos, respectively. The protein content of day 7 zona‐enclosed blastocysts was 337 ± 58.0 ng embryo‐1. These values suggest that prior to compaction and blastulation, the early cleavage stage bovine embryo has a higher rate of protein degradation than that of synthesis. Net growth is observed only after initiation of compaction. The protein content of day 7 blastocysts was measured in embryos following in vitro production and culture in the same media supplemented with either 0.5% w/v polyvinyl alcohol (PVA), 8 mg ml‐1 BSA, 8 mg ml‐1 BSA and further supplemented with 10% fetal calf serum (FCS) from the beginning of culture (FCS‐D1), 8 mg ml‐1 BSA and 10% FCS from the fourth day of culture (day 5 of development) or from in vivo‐derived day 7 blastocysts. Protein content was significantly (P< 0.05) lower in PVA‐cultured embryos than other treatments. To determine if this difference in PVA‐cultured embryos was due to a difference in the rate of protein synthesis, comparisons were made between day 7 embryos derived from BSA‐culture and either PVA‐culture, FCS‐D1 culture or in vivo‐derived embryos. Despite differences in diameter, no significant difference was observed in the incorporation of L‐[2,3,4,5,6‐3H]‐phenylalanine into the TCA‐precipitable fraction in any of the three comparisons made. However, incubation in the presence of FITC‐labelled BSA or β‐casein and examination under either fluorescence or confocal microscopy revealed that protein in the extra‐embryonic environment was actively taken up by the trophectoderm of day 7 blastocysts, most likely by endocytosis. These results suggest that exogenous protein is an important nutritive source, probably maintaining intracellular amino acid pools. Results obtained from the production of embryos in protein‐free medium should be viewed with the knowledge that such embryos differ metabolically from those embryos grown in the presence of protein, including in vivo‐derived embryos. Mol. Reprod. Dev. 50:139–145, 1998. © 1998 Wiley‐Liss, Inc.